Abstract Significance: This research will develop the Trojan horse liposome (THL) platform technology for delivery to organs in vivo of non-viral plasmid DNA therapeutics. THLs, in combination with tissue-specific gene promoters, enable the expression of the therapeutic gene in distant sites of the body, including the brain following a non-invasive intravenous administration. The technology is particularly suited to therapeutic DNA delivery to brain across the blood-brain barrier (BBB). The THL platform can deliver an unlimited number of therapeutic genes for treatment of monogenic diseases, such as severe inborn errors of metabolism and orphan diseases, or inherited blindness, as well as polygenic disease such as neurodegeneration. THLs can deliver plasmid DNA encoding short hairpin RNA to enable RNA interference. Hypothesis: THLs have been investigated previously, but only at the microscale R&D level. The present research plan is based on the hypothesis that THL manufacturing can be scaled by orders of magnitude, from the R&D stage, and that manufactured THLs will have an acceptable shelf life to enable commercialization. This is achievable with the combined use of a pressurized stainless steel extruder and re-formulation of THLs as a lyophilized freeze-dried preparation. THLs made with this new manufacturing model will be reduced to practice with a `use case,' which will treat the mouse model of Krabbe disease, a lysosomal storage disease caused by a missense mutation in the gene encoding the galactosylceramidase (GALC) lysosomal enzyme. Preliminary Data: Prior work has shown that THLs can be targeted to brain with monoclonal antibodies (MAb) against either the insulin receptor or the transferrin receptor (TfR) for delivery of plasmid DNA to either the monkey brain or the mouse brain, including mouse models of lysosomal storage disease. Specific Aims (Phase I): (1) A plasmid DNA encoding the human GALC enzyme will be genetically engineered under the influence of a 1.6 kb gene promoter, in parallel with production of the monoclonal antibody (MAb) Trojan horse. (2) THLs targeted with the monoclonal antibody and encapsulated with the GALC plasmid DNA will be produced with a large scale extruder device suitable for production of clinical trial quantities of THLs. (3) The biologic activity of the monoclonal antibody- targeted THLs, encapsulating the GALC plasmid DNA, will be confirmed in human Krabbe disease fibroblasts, with both immune detection of the GALC enzyme, as well as measurements of intracellular GALC enzyme activity. Specific Aims (Phase II): The Manufacturing Module is comprised of: (1) Genetic engineering of a humanized form of the targeting MAb and 100 mg gigapreps of low endotoxin GALC plasmid DNA; (2) MAb production from either myeloma cells or stably transfected CHO cells; (3) Large-scale THL production with the pressurized extruder and a programmed freeze-dry process. The Preclinical Module is comprised of (1) A collaboration with The Jackson Lab for treatment of a twitcher missense mouse model of Krabbe disease; (2) A collaboration with a CRO for execution of a 4-week dose-ranging study of THLs in the non-human primate.